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Biomedical subjects

J A Gallant

Publications and source records attributed to J A Gallant.

6 recordsLinked to original sources
PubMed1992

Leftward ribosome frameshifting at a hungry codon.

Previous experiments have shown that limitation for certain aminoacyl-tRNA species results in phenotypic suppression of a subset of frameshift mutant alleles, including members in both the (+) and (-) incorrect reading frames. Here, we demonstrate that such phenotypic suppression can occur through a ribosome reading frame shift at a hungry AAG codon calling for lysyl-tRNA in short supply. Direct amino acid sequence analysis of the product and DNA sequence manipulation of the gene demonstrate that the ribosome frameshift occurs through a movement of one base to the left, so as to decode the triplet overlapping the hungry codon from the left or 5' side, followed by continued normal translation in the new, shifted reading frame.

Amino Acid Sequence↗
PubMed1992

Context rules of rightward overlapping reading.

We have investigated the mechanism and sequence context rules governing ribosome frameshifting promoted by aminoacyl-tRNA limitation. In the case of one shifty sequence, frameshifting promoted by lysyl-tRNA limitation occurs at the sequence AAG C and is due to rightward movement of the ribosome so as to read the AGC triplet overlapping the hungry codon from the right. The frequency of this event is unaffected by sequence elements more than three bases to the left (upstream) or two bases to the right (downstream) of the hungry codon, and only slightly affected by the identity of the base two bases to the right. It is strongly affected by the base immediately to the right of the hungry codon, which becomes the wobble base of the shifted triplet; and by the third base of the hungry codon, even though the two synonyms (AAG and AAA) call for the same aminoacyl-tRNA; and by the identity of the base immediately to the left of the hungry codon. The latter result suggests that the aminoacyl-tRNA in the P site affects the maintenance of reading frame at the adjacent A site of the ribosome. However, the DNA sequence makes it seem unlikely that the P-site tRNA shifts to the right in concert with the A-site tRNA, a mechanism that can account for leftward frameshifting (in the opposite direction) in retroviral translation. The specificity of sequence determinants of leftwing versus rightwing frameshifting is discussed.

Amino Acid Sequence↗
PubMed1991

The role of RNA polymerase in transcriptional fidelity.

The overall transcription of DNA has previously been demonstrated to proceed at extremely high levels of accuracy. We review the evidence that the process of transcription is subject to proof-reading in the Hopfield sense. In addition, we speculate that the proof-reading activity associated with transcription is subject to cyclical phase transitions. That is, during periods of low processivity associated with initiation, RNA synthesis is relatively imprecise. The transition to the elongation phase of RNA synthesis, characterized by a shift to high processivity, is accompanied by enhanced proof-reading. A model for the damping of transcriptional errors, based on a PPi-mediated processive pyrophosphorolysis reaction, is discussed in terms of pausing during transcription.

Bacterial Proteins↗
PubMed1989

Transcriptional proofreading in Escherichia coli.

A novel transcriptional proofreading mechanism associated with the beta-subunit of wild-type RNA polymerase from Escherichia coli is suggested from the following data. The purified holoenzyme contains an NTPase activity which specifically converts noncognate NTPs to their corresponding NDP in a template-dependent manner during in vitro transcription of synthetic single- and double-stranded templates. In contrast, purified enzyme from an rpoB mutant which shows increased transcriptional error lacked template-dependent NTP hydrolytic activity. The NTP hydrolytic activity of wild-type enzyme was critically dependent on the integrity of the initiation complex, and required continued transcriptional elongation. Transcription and translation of the lacZ gene proceeded 17% faster in the mutant than in its wild-type parent. These results are discussed in terms of a proofreading model in which the rate of transcription is limited by proofreading events that involve recognition and hydrolysis of noncognate NTPs before they can be misincorporated into RNA.

DNA-Directed RNA Polymerases↗